ribG

ABSTRACT

The invention provides ribG polypeptides and polynucleotides encoding ribG polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ribG polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

[0001] This application claims benefit of PCT Application, NumberPCT/US97/02318, filed Feb. 19, 1997.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to novel polynucleotides and polypeptides of the riboflavinspecific deaminase family, hereinafter referred to as “ribG”.

BACKGROUND OF THE INVENTION

[0003] It is particularly preferred to employ Staphylococcal genes andgene products as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

[0004] Riboflavin (vitamin B2) is a member of the B complex of vitaminswhich function as coenzymes in metabolic reactions. Riboflavin has twocoenzyme forms, flavin mononucleotide (FMN) and flavin adeninedinucleotide (FAD) which act in oxidation-reduction reactions such asthe cytochrome system of electron transport and the oxidativedegradation of pyruvate, fatty acids and amino acids. The firstcommitted step in the biosynthesis of riboflavin is the opening of theimidazole ring of GTP. In the presence of 3 H₂O and Mg⁺⁺, the C-8 of GTPis released as formate accompanied by the release of pyrophosphate bythe action of GTP cyclohyrolase II (GCH2; EC 3.5.4.25). This enzymefunction is encoded by ribA in bacteria and rib1 in yeast species.Through a series of steps, involving 3,4-dihydroxy-2-butanone 4phosphate synthase (ribA), 6,7-dimethyl-8-ribityllumazine synthase(ribH), riboflavin synthase (ribB), pyrimidine deaminase and pyrimidinereductase (ribG), enzymes encoded by genes within the riboflavinbiosynthesis operon, riboflavin is formed. Because the genes requiredfor riboflavin biosynthesis are present in many pathogenicmicroorganisms, and since riboflavin biosynthesis has shown to berequired for virulence in the swine pathogen Actinobacilluspleuropneumoniae (Fuller, T E, et al. (1996) A riboflavin auxotroph ofActinobacillus pleuropneumoniae is attenuated in swine. Infect. Immun.64:4659-4664), these gene products represent broad spectrumantibacterial as well as antifungal targets.

[0005] The frequency of Staphylococcus aureus infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Staphylococcus aureus strains which are resistant tosome or all of the standard antibiotics. This phenomenon has created ademand for both new anti-microbial agents, vaccines, and diagnostictests for this organism.

[0006] Clearly, there exists a need for factors, such as the nbGembodiments of the invention, that have a present benefit of beinguseful to screen compounds for antibiotic activity. Such factors arealso useful to determine their role in pathogenesis of infection,dysfunction and disease. There is also a need for identification andcharacterization of such factors and their antagonists and agonists tofind ways to prevent, ameliorate or correct such infection, dysfunctionand disease.

[0007] Certain of the polypeptides of the invention possess amino acidsequence homology to a known Bacillus subtilis ribG protein.

SUMMARY OF THE INVENTION

[0008] It is an object of the invention to provide polypeptides thathave been identified as novel ribG polypeptides by homology between theamino acid sequence set out in Table 1 [SEQ ID NO: 2 or 4] and a knownamino acid sequence or sequences of other proteins such as Bacillussubtilis ribG protein.

[0009] It is a further object of the invention to providepolynucleotides that encode ribG polypeptides, particularlypolynucleotides that encode the polypeptide herein designated ribG.

[0010] In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding ribG polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO: 1 or 3] which includes a fulllength gene, or a variant thereof.

[0011] In another particularly preferred embodiment of the inventionthere is a novel ribG protein from Staphylococcus aureus comprising theamino acid sequence of Table 1 [SEQ ID NO:2 or 4], or a variant thereof.

[0012] In accordance with another aspect of the invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressible by the Staphylococcus aureus WCUH29 on deposit strain, whichnucleic acid is contained in the deposited strain.

[0013] A further aspect of the invention there are provided isolatednucleic acid molecules encoding ribG, particularly Staphylococcus aureusribG, including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

[0014] In accordance with another aspect of the invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of ribG and polypeptides encoded thereby.

[0015] Another aspect of the invention there are provided novelpolypeptides of Staphylococcus aureus referred to herein as ribG as wellas biologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

[0016] Among the particularly preferred embodiments of the invention arevariants of ribG polypeptide encoded by naturally occurring alleles ofthe ribG gene.

[0017] In a preferred embodiment of the invention there are providedmethods for producing the aforementioned ribG polypeptides.

[0018] In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

[0019] In accordance with certain preferred embodiments of theinvention, there are provided products, compositions and methods forassessing ribG expression, treating disease, assaying genetic variation,and administering a ribG polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStaphylococcus aureus bacteria.

[0020] In accordance with certain preferred embodiments of this andother aspects of the invention there are provided polynucleotides thathybridize to ribG polynucleotide sequences, particularly under stringentconditions.

[0021] In certain preferred embodiments of the invention there areprovided antibodies against ribG polypeptides.

[0022] In other embodiments of the invention there are provided methodsfor identifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

[0023] In accordance with yet another aspect of the invention, there areprovided ribg agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

[0024] In a further aspect of the invention there are providedcompositions comprising a ribG polynucleotide or a ribG polypeptide foradministration to a cell or to a multicellular organism.

[0025] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following descriptions and from reading theother-parts of the present disclosure

DESCRIPTION OF THE INVENTION

[0026] The invention relates to novel ribG polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novel ribG ofStaphylococcus aureus, which is related by amino acid sequence homologyto Bacillus subtilis ribG polypeptide. The invention relates especiallyto ribG having the nucleotide and amino acid sequences set out in Table1 as SEQ ID NO: 1 and SEQ ID NO: 2 respectivelyribG.

Table 1

[0027] ribG Polynucleotide and Polypeptide Sequences

[0028] (A) Sequences from Staphylococcus aureus ribG PolynucleotideSequence [SEQ ID NO: 1].ATGGATTATGCGATTCAACTTGCAAATATGGTACAAGGTCAAACAGGTGTTAATCCACCCGTTGGCGCTGTTGTAGTTAATGAAGGTAGGATTGTTGGTATTGGTGCACACTTGAGAAAAGGTGACAAGCATGCGGAGGTTCAAGCACTTGATATGGCACAACAAAATGCTGAAGGTGCGACGATTTATATTACGTTAGAGCCATGTAGTCATTTTGGTTCAACACCACCCTGTGTTAACAAAATTATTGATTGTAAGATAGCAAAAGTAGTATACGCAACAAAAGACAATTCGTTAGACACACATGGTGATGAGACGTTACGGGCTCACGGTATTGAGGTTGAATGCGTTGATGATGAACGGGCATCACAATTATACCAAGACTTTTTTAAAGCAAAAGCAAAGCAACTGCCACAAATTACAGTGAAAGTATCTGCAAGTTTAGATGGTAAACAAGCGAATGATAATGGACAAAGTCAATGGATTACTAACAAAGAGGTTAAACAAGATGTCTATAAGTTAAGACATCGACACGACGCAGTGTTAACTGGAAGACGTACAGTTGAATTAGATGATCCACAATATACTACACGTATTCAAGATGGAAAAAACCCTATAAAAGTAATATTGTCTAAGTCTGGGAATATTCATTTTAATCAGCAAATTTATCAAGATGAATCAACACCAATTTGGATATATACTGAAAATCCAAATTTAACAAGCAATCAAACACATATTGAAATTATTTACTTGAAGTCTTGTGATTTAACAACAATTCTTCACAATTTATATAAAAGAGGAGTTGGAACTTTGCTAGTCGAGGCAGGTCCAACCACTACTTCAGAATTCTCCATCTATTATATAGATGAATTTATTCTCTATTATGCCCCGAAATTAATTGGCGGATCTGGAAATTATCAATTTTATCAAACAAATGATGTGATTGAGATACCAGATGCGAACCAATTTGAAATTGTTCATTCCGAGTTATTAAATCAAAATGTTAAATTAACTTTACGAAAGAAGTGA-3′

[0029] (B) Staphylococcus aureus ribG Polypeptide Sequence Deduced fromthe Polynucleotide Sequence in this Table [SEQ ID NO:2]. NH₂-MDYAIQLANMVQGQTGVNPPVGAVVVNEGRIVGIGAHLRKGDKHAEVQALDMAQQNAEGATIYITLEPCSHFGSTPPCVNKIIDCKIAKVVYATKDNSLDTHGDETLPAHGIEVECVDDEPASQLYQDFFKAKAKQLPQITVKVSASLDGKQANDNGQSQWITNKEVKQDVYKLRHRHDAVLTGRRTVELDDPQYTTRIQDGKNPIKVILSKSGNIHFNQQIYQDESTPIWIYTENPNLTSNQTHIEIIYLKSCDLTTILHNLYKRGVGTLLVEAGPTTTSEFSIYYIDEFILYYAPKLIGGSGNYQFYQTNDVIEIPDANQFEIVHSELLNQNVKLTLRKK-COOH

[0030] (C) Polynucleotide Sequences Comprising Staphylococcus aureusribG ORF Sequence [SEQ ID NO:3]. 5′-AANCACCAATCCNATTGGGAGGNAATCCAAATCAATNCCCGGANNCCCAATCCAAGTTAATTAAGTCCAAGGTTTTGGAACATTACCAAATATGATTCCGATGAGGTCAAATGNCAANCGGTGTTAATAAACTACGAAATGNTGTGNAAATGATAGTAGANCAAGTTGCGCATACAGTNTCTCNATTATATGATGCTTTAGAATCGAATGAGCAACAACAGCGCAGTTACAATCAATAATTTGTAACTAGAAGATAATAAAGAGAACGCTCTATAGAGACGAATTGAAGGTTTGATTTTAATGTCTGTTAGTAAGAATCATATCAATGAGATGCCTATAGTACTCAGATTATATTAAATTAAAACCGTCATTAATTGTTTTTTTAGAAAACATATAGTATCATTTTAAATGTAGTTGACATACTACGTACTCAAATAATCTATAACAATTTCATATATAATTCTTTCGGGGCAGGGTGAAATTCCCAACCGGCAGTAAATAAAGCCTGCGACCTGCTAATATGTTTCATATTAGTGGCTGATCTAGTGAGATTCTAGAGCCGACAGTTAAAGTCTGGATGGGAGAAAGAATGTTAATTATCGACAAAGATAATGTAGCGTATTTGTAAAAATGTGTACAAATAGGCTTATTTAACGATAAATTTTTCTCCTTTGCATCTTAATTCATGATGTGAGGATTTTTTGTTTATAGAGGTGATCATTTGAGTCAATTTATGGATTATGCGATTCAACTTCCAAATATGGTACAAGGTCANACAGGTGTTAATCCACCCGTTGGCGCTGTTGTAGTTAATGAAGGTAGGATTGTTGGTATTGGTGCACACTTGAGAAAAGGTGACAAGCATGCGGAGGTTCAAGCACTTGATATGGCACAACANAATGCTGAAGGTGCGACGATTTATATTACGTTAGAGCCATGTAGTCATTTTGGTTCAACACCACCCTGTGTTAACAAAATTATTGATTGTAAGATAGCANAAGTAGTATTACNCAACANAAGACAATTCCGTTAGACACACATGGGTGATGAGACGTTACGGGGCTCCACGGTATTTGAGGGTTGAATTGCGTTGGATGATGAACGGGCATCACAATTATACCAAAGACTTTTTTTAAAGCAAAAAGCAAAGCAACTTGCCACAAAATTACAGTGAAAGTNTCTTGAAAGTTTAGATGGGTAAACAAAGCGAATTGATAATGGACAAAGTCAATGGATTACTAACAAAGAGGTTAAACAAGATGTCTATAG-3′

[0031] (D) Staphylococcus aureus ribG Polypeptide Sequence Deduced fromthe Polynucleotide ORF Sequence in this Table [SEQ ID NO:4]. NH₂-MDYAIQLPNMVQGXTGVNPPVGAVVVNEGRIVGIGAHLRKGDKHAEVQALDMAQXNAEGATIYITLEPCSHFGSTPPCVNKIIDCKIAXVVLXNXRQFR —COOH

[0032] Deposited Materials

[0033] A deposit containing a Staphylococcus aureus WCUH 29 strain hasbeen deposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on 11 Sep. 1995 and assigned NCIMB Deposit No. 40771, andreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

[0034] The deposited strain contains the full length ribG gene. Thesequence of the polynucleotides contained in the deposited strain, aswell as the amino acid sequence of the polypeptide encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein.

[0035] The deposit of the deposited strain has been made under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

[0036] A license may be required to make, use or sell the depositedstrain, and compounds derived therefrom, and no such license is herebygranted.

[0037] One aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStaphylococcus aureus WCUH 29 strain contained in the deposited strain.Further provided by the invention are ribG nucleotide sequences of theDNA in the deposited strain and amino acid sequences encoded thereby.Also provided by the invention are ribG polypeptide sequences isolatedfrom the deposited strain and amino acid sequences derived therefrom.

[0038] Polypeptides

[0039] The polypeptides of the invention include a polypeptide of Table1 [SEQ ID NO:2 or 4] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of ribG, and also those which have at least 70% identity to apolypeptide of Table 1 [SEQ ID NO:1 or 3]or the relevant portion,preferably at least 80% identity to a polypeptide of Table 1 [SEQ IDNO:2 or 4and more preferably at least 90% similarity (more preferably atleast 90% identity) to a polypeptide of Table 1 [SEQ ID NO:2 or 4] andstill more preferably at least 95% similarity (still more preferably atleast 95% identity) to a polypeptide of Table 1 [SEQ ID NO:2 or 4] andalso include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

[0040] The invention also includes polypeptides of the formula:

—X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

[0041] wherein, at the amino terminus, X is hydrogen, and at thecarboxyl terminus, Y is hydrogen or a metal, R₁ and R₃ are any aminoacid residue, m is an integer between 1 and 1000 or zero, n is aninteger between 1 and 1000 or zero, and R₂ is an amino acid sequence ofthe invention, particularly an amino acid sequence selected fromTable 1. In the formula above R₂ is oriented so that its amino terminalresidue is at the left, bound to R_(1,) and its carboxy terminal residueis at the right, bound to R₃. Any stretch of amino acid residues denotedby either R group, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer.

[0042] A fragment is a variant polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned polypeptides. As with ribG polypeptides fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

[0043] Preferred fragments include, for example, truncation polypeptideshaving a portion of an amino acid sequence of Table 1 [SEQ ID NO:2 or4], or of variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Staphylococcus aureus, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

[0044] Also preferred are biologically active fragments which are thosefragments that mediate activities of ribG, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

[0045] Variants that are fragments of the polypeptides of the inventionmay be employed for producing the corresponding full-length polypeptideby peptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

[0046] In addition to the standard single and triple letterrepresentations for amino acids, the term “X” or “Xaa” may also be usedin describing certain polypeptides of the invention. “X” and “Xaa” meanthat any of the twenty naturally occuring amino acids may appear at sucha designated position in the polypeptide sequence.

[0047] Polynucleotides

[0048] Another aspect of the invention relates to isolatedpolynucleotides, including the full length gene, that encode the ribGpolypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2or 4] and polynucleotides closely related thereto and variants thereof.

[0049] Using the information provided herein, such as a polynucleotidesequence set out in Table 1 [SEQ ID NO:1 or 3], a polynucleotide of theinvention encoding ribG polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH29 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 (SEQ ID NO: 1 or 3],typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E.coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 [SEQ ID NO:1 or 3] was discovered in a DNA libraryderived from Staphylococcus aureus WCUH 29.

[0050] The DNA sequence set out in Table 1 [SEQ ID NO:1 or 3] containsan open reading frame encoding a protein having about the number ofamino acid residues set forth in Table 1 [SEQ ID NO:2 or 4] with adeduced molecular weight that can be calculated using amino acid residuemolecular weight values well known in the art. The polynucleotide of SEQID NO: 1, between nucleotide number I and the stop codon which begins atnucleotide number 1027 of SEQ ID NO: 1, encodes the polypeptide of SEQID NO:2.

[0051] RibG of the invention is structurally related to other proteinsof the riboflavin specific deaminase family.

[0052] The invention provides a polynucleotide sequence identical overits entire length to a coding sequence in Table 1 [SEQ ID NO: 1 or 3].Also provided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro-protein sequence. The polynucleotidemay also contain non-coding sequences, including for example, but notlimited to non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences, termination signals, ribosome binding sites,sequences that stabilize mRNA, introns, polyadenylation signals, andadditional coding sequence which encode additional amino acids.

[0053] For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

[0054] A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 1 to the nucleotide immediately upstream of orincluding nucleotide 1027 set forth in SEQ ID NO: 1 of Table 1, both ofwhich encode the ribG polypeptide.

[0055] The invention also includes polynucleotides of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

[0056] wherein, at the 5′ end of the molecule, X is hydrogen, and at the3′ end of the molecule, Y is hydrogen or a metal, R₁ and R₃ is anynucleic acid residue, m is an integer between 1 and 3000 or zero, n isan integer between 1 and 3000 or zero, and R₂ is a nucleic acid sequenceof the invention, particularly a nucleic acid sequence selected fromTable 1. In the polynucleotide formula above R₂ is oriented so that its5′ end residue is at the left, bound to R₁, and its 3′ end residue is atthe right, bound to R₃. Any stretch of nucleic acid residues denoted byeither R group, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer. In apreferred embodiment m and/or n is an integer between 1 and 1000.

[0057] It is most preferred that the polynucleotides of the inventionsare derived from Staphylococcus aureus, however, they may preferably beobtained from organisms of the same taxonomic genus. They may also beobtained, for example, from organisms of the same taxonomic family ororder.

[0058] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus ribG havingan amino acid sequence set out in Table 1 [SEQ ID NO:2 or 4]. The termalso encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,interrupted by integrated phage or an insertion sequence or editing)together with additional regions, that also may contain coding and/ornon-coding sequences.

[0059] The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having adeduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4]. Variants thatare fragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

[0060] Further particularly preferred embodiments are polynucleotidesencoding ribG variants, that have the amino acid sequence of ribGpolypeptide of Table 1 [SEQ ID NO:2 or 4] in which several, a few, 5 to10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted,deleted or added, in any combination. Especially preferred among theseare silent substitutions, additions and deletions, that do not alter theproperties and activities of ribG.

[0061] Further preferred embodiments of the invention arepolynucleotides that are at least 70% identical over their entire lengthto a polynucleotide encoding ribG polypeptide having an amino acidsequence set out in Table 1 [SEQ ID NO:2 or 4], and polynucleotides thatare complementary to such polynucleotides. Alternatively, most highlypreferred are polynucleotides that comprise a region that is at least80% identical over its entire length to a polynucleotide encoding ribGpolypeptide and polynucleotides complementary thereto. In this regard,polynucleotides at least 90% identical over their entire length to thesame are particularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

[0062] Preferred embodiments are polynucleotides that encodepolypeptides that retain substantially the same biological function oractivity as the mature polypeptide encoded by a DNA of Table 1 [SEQ IDNO: 1 or 3].

[0063] The invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in0.1×SSC at about 65° C. Hybridization and wash conditions are well knownand exemplified in Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein.

[0064] The invention also provides a polynucleotide consistingessentially of a polynucleotide sequence obtainable by screening anappropriate library containing the complete gene for a polynucleotidesequence set forth in SEQ ID NO:1 under stringent hybridizationconditions with a probe having the sequence of said polynucleotidesequence set forth in SEQ ID NO: 1 or a fragment thereof; and isolatingsaid DNA sequence. Fragments useful for obtaining such a polynucleotideinclude, for example, probes and primers described elsewhere herein.

[0065] As discussed additionally herein regarding polynucleotide assaysof the invention, for instance, polynucleotides of the invention asdiscussed above, may be used as a hybridization probe for RNA, cDNA andgenomic DNA to isolate full-length cDNAs and genomic clones encodingribG and to isolate cDNA and genomic clones of other genes that have ahigh sequence similarity to the ribG gene. Such probes generally willcomprise at least 15 bases. Preferably, such probes will have at least30 bases and may have at least 50 bases. Particularly preferred probeswill have at least 30 bases and will have 50 bases or less.

[0066] For example, the coding region of the ribG gene may be isolatedby screening using a DNA sequence provided in Table 1 [SEQ ID NO: 1 or3] to synthesize an oligonucleotide probe. A labeled oligonucleotidehaving a sequence complementary to that of a gene of the invention isthen used to screen a library of cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0067] The polynucleotides and polypeptides of the invention may beemployed, for example, as research reagents and materials for discoveryof treatments of and diagnostics for disease, particularly humandisease, as further discussed herein relating to polynucleotide assays.

[0068] Polynucleotides of the invention that are oligonucleotidesderived from the sequences of Table 1 [SEQ ID NOS: 1 or 2 or 3 or 4] maybe used in the processes herein as described, but preferably for PCR, todetermine whether or not the polynucleotides identified herein in wholeor in part are transcribed in bacteria in infected tissue. It isrecognized that such sequences will also have utility in diagnosis ofthe stage of infection and type of infection the pathogen has attained.

[0069] The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed-away fromthe mature protein by cellular enzymes.

[0070] A precursor protein, having the mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proprotein's.

[0071] In addition to the standard A, G, C, T/U representations fornucleic acid bases, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA bases may appear at such a designated position in the DNA or RNAsequence, except it is preferred that N is not a base that when taken incombination with adjacent nucleotide positions, when read in the correctreading frame, would have the effect of generating a prematuretermination codon in such reading frame.

[0072] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

[0073] Vectors, Host Cells, Expression

[0074] The invention also relates to vectors that comprise apolynucleotide or polynucleotides of the invention, host cells that aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

[0075] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof orpolynucleotides of the invention. Introduction of a polynucleotide intothe host cell can be effected by methods described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), such as, calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction and infection.

[0076] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, enterococci E. coli,streptomyces and Bacillus subtilis cells; fungal cells, such as yeastcells and Aspergillus cells; insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C1 27, 3T3,BHK, 293 and Bowes melanoma cells; and plant cells.

[0077] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

[0078] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0079] Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

[0080] Diagnostic Assays

[0081] This invention is also related to the use of the ribGpolynucleotides of the invention for use as diagnostic reagents.Detection of ribG in a eukaryote, particularly a mammal, and especiallya human, will provide a diagnostic method for diagnosis of a disease.Eukaryotes (herein also “individual(s)”), particularly mammals, andespecially humans, particularly those infected or suspected to beinfected with an organism comprising the ribG gene may be detected atthe nucleic acid level by a variety of techniques.

[0082] Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA, cDNA and genomic DNA may also be used in thesame ways. Using amplification, characterization of the species andstrain of prokaryote present in an individual, may be made by ananalysis of the genotype of the prokaryote gene. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the genotype of a reference sequence. Point mutationscan be identified by hybridizing amplified DNA to labeled ribGpolynucleotide sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase digestion or bydifferences in melting temperatures. DNA sequence differences may alsobe detected by alterations in the electrophoretic mobility of the DNAfragments in gels, with or without denaturing agents, or by direct DNAsequencing. See, e.g., Myers et al., Science, 230: 1242 (1985). Sequencechanges at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or a chemicalcleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA,85: 43974401 (1985).

[0083] Cells carrying mutations or polymorphisms in the gene of theinvention may also be detected at the DNA level by a variety oftechniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations. It is particularly preferred to usedRT-PCR in conjunction with automated detection systems, such as, forexample, GeneScan. RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR or RT-PCR. As an example, PCR primers complementary toa nucleic acid encoding ribG can be used to identify and analyzemutations. Examples of representative primers are shown below in Table2. TABLE 2 Primers for amplification of ribG polynucleotides SEQ ID NOPRIMER SEQUENCE 5 5′-ATGAAGGTAGGATTGTTGGTA-3′ 65′-AGTCTTGGTATAATTGTGATGC-3′

[0084] The invention also includes primers of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

[0085] wherein, at the 5′ end of the molecule, X is hydrogen, and at the3′ end of the molecule, Y is hydrogen or a metal, R₁ and R₃ is anynucleic acid residue, m is an integer between 1 and 20 or zero , n is aninteger between 1 and 20 or zero, and R₂ is a primer sequence of theinvention, particularly a primer sequence selected from Table 2. In thepolynucleotide formula above R₂ is oriented so that its 5′ end residueis at the left, bound to R₁, and its 3′ end residue is at the right,bound to R₃. Any stretch of nucleic acid residues denoted by either Rgroup, where m and/or n is greater than 1, may be either a heteropolymeror a homopolymer, preferably a heteropolymer being complementary to aregion of a polynucleotide of Table 1. In a preferred embodiment mand/or n is an integer between 1 and 10.

[0086] The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifying ribG DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

[0087] The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, comprising determining from a sample derived froman individual a increased level of expression of polynucleotide having asequence of Table 1 [SEQ ID NO: 1 or 3]. Increased or decreasedexpression of ribG polynucleotide can be measured using any on of themethods well known in the art for the quantation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods.

[0088] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of ribG protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a ribGprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

[0089] Antibodies

[0090] The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

[0091] Antibodies generated against the polypeptides of the inventioncan be obtained by administering the polypeptides or epitope-bearingfragments, analogues or cells to an animal, preferably a nonhuman, usingroutine protocols. For preparation of monoclonal antibodies, anytechnique known in the art that provides antibodies produced bycontinuous cell line cultures can be used. Examples include varioustechniques, such as those in Kohler, G. and Milstein, C., Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole etal., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985).

[0092] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

[0093] Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-ribG or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

[0094] If two antigen binding domains are present each domain may bedirected against a different epitope—termed ‘bispecific’ antibodies.

[0095] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptides to purify the polypeptidesby affinity chromatography.

[0096] Thus, among others, antibodies against ribG-polypeptide may beemployed to treat infections, particularly bacterial infections.

[0097] Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

[0098] The polypeptide, such as an antigenically or immunologicallyequivalent derivative or a fusion protein thereof is used as an antigento immunize a mouse or other animal such as a rat or chicken. The fusionprotein may provide stability to the polypeptide. The antigen may beassociated, for example by conjugation, with an immunogenic carrierprotein for example bovine serum albumin (BSA) or keyhole limpethaemocyanin (KLH). Alternatively a multiple antigenic peptide comprisingmultiple copies of the protein or polypeptide, or an antigenically orimmunologically equivalent polypeptide thereof may be sufficientlyantigenic to improve immunogenicity so as to obviate the use of acarrier.

[0099] Preferably, the antibody or variant thereof is modified to makeit less immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

[0100] The use of a polynucleotide of the invention in geneticimmunization will preferably employ a suitable delivery method such asdirect injection of plasmid DNA into muscles (Wolff et al., Hum MolGenet 1992, 1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419),delivery of DNA complexed with specific protein carriers (Wu et al., JBiol Chem. 1989: 264,16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA, 1986:83,9551), encapsulationof DNA in various forms of liposomes (Kaneda et al., Science1989:243,375), particle bombardment (Tang et al., Nature 1992, 356:152,Eisenbraun et al., DNA Cell Biol 1993, 12:79 1) and in vivo infectionusing cloned retroviral vectors (Seeger et al., PNAS USA 1984:81,5849).

[0101] Antagonists and Agonists—Assays and Molecules

[0102] Polypeptides of the invention may also be used to assess thebinding of small molecule substrates and ligands in, for example, cells,cell-free preparations, chemical libraries, and natural productmixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics. See, e.g., Coliganet al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

[0103] The invention also provides a method of screening compounds toidentify those which enhance (agonist) or block (antagonist) the actionof ribG polypeptides or polynucleotides, particularly those compoundsthat are bacteriostatic and/or bacteriocidal. The method of screeningmay involve high-throughput techniques. For example, to screen foragonists or antagoists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising ribG polypeptide and a labeledsubstrate or ligand of such polypeptide is incubated in the absence orthe presence of a candidate molecule that may be a ribg agonist orantagonist. The ability of the candidate molecule to agonize orantagonize the ribG polypeptide is reflected in decreased binding of thelabeled ligand or decreased production of product from such substrate.Molecules that bind gratuitously, ie., without inducing the effects ofribG polypeptide are most likely to be good antagonists. Molecules thatbind well and increase the rate of product production from substrate areagonists. Detection of the rate or level of production of product fromsubstrate may be enhanced by using a reporter system. Reporter systemsthat may be useful in this regard include but are not limited tocolorimetric labeled substrate converted into product, a reporter genethat is resporisive to changes in ribG polynucleotide or polypeptideactivity, and binding assays known in the art.

[0104] Another example of an assay for ribG antagonists is a competitiveassay that combines ribG and a potential antagonist with ribG-bindingmolecules, recombinant ribG binding molecules, natural substrates orligands, or, substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. RibG can be labeled, such as byradioactivity or a calorimetric compound, such that the number of ribGmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

[0105] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing ribG-induced activities, thereby preventing the action of ribGby excluding ribg from binding.

[0106] Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, JNeurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of ribG.

[0107] Each of the DNA sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the DNA sequences encoding the aminoterminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

[0108] The invention also provides the use of the polypeptide,polynucleotide or inhibitor of the invention to interfere with theinitial physical interaction between pathogen and mammalian hostresponsible for sequelae of infection. In particular the molecules ofthe invention may be used: in the prevention of adhesion of bacteria, inparticular gram positive bacteria, to mammalian extracellular matrixproteins on in-dwelling devices or to extracellular matrix proteins inwounds; to block ribG protein-mediated mammalian cell invasion by, forexample, initiating phosphorylation of mammalian tyrosine kinases(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block bacterialadhesion between mammalian extracellular matrix proteins and bacterialribG proteins that mediate tissue damage and; to block the normalprogression of pathogenesis in infections initiated other than by theimplantation of in-dwelling devices or by other surgical techniques.

[0109] The antagonists and agonists: of the invention may be employed,for instance, to inhibit and treat diseases.

[0110]Helicobacter pylori (herein H. pylori) bacteria infect thestomachs of over one-third of the world's population causing stomachcancer, ulcers, and gastritis (International Agency for Research onCancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori(International Agency for Research on Cancer, Lyon, France;http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylon and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofribG) found using screens provided by the invention, particularlybroad-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also cure gastric ulcers and gastritis.

[0111] Vaccines

[0112] Another aspect of the invention relates to a method for inducingan immunological response in an individual, particularly a mammal whichcomprises inoculating the individual with ribG, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Staphylococcus aureus infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector to direct expression of ribG, or afragment or a variant thereof, for expressing ribG, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/ or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise. Such nucleic acid vector may comprise DNA, RNA,a modified nucleic acid, or a DNA/RNA hybrid.

[0113] A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a ribG or protein coded therefrom,wherein the composition comprises a recombinant ribG or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid ribG or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

[0114] A ribG polypeptide or a fragment thereof may be fused withco-protein which may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

[0115] Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

[0116] Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

[0117] The polypeptide may be used as an antigen for vaccination of ahost to produce specific antibodies which protect against invasion ofbacteria, for example by blocking adherence of bacteria to damagedtissue. Examples of tissue damage include wounds in skin or connectivetissue caused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

[0118] The invention also includes a vaccine formulation which comprisesan immunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

[0119] While the invention has been described with reference to certainribg protein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

[0120] Compositions, Kits and Administration

[0121] The invention also relates to compositions comprising thepolynucleotide or the polypeptides discussed above or their agonists orantagonists. The polypeptides of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration. Theinvention further relates to diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

[0122] Polypeptides and other compounds of the invention may be employedalone or in conjunction with other compounds, such as therapeuticcompounds.

[0123] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0124] In therapy or as a prophylactic, the active agent may beadministered to an individual as an injectable composition, for exampleas a sterile aqueous dispersion, preferably isotonic.

[0125] Alternatively the composition may be formulated for topicalapplication for example in the form of ointments, creams, lotions, eyeointments, eye drops, ear drops, mouthwash, impregnated dressings andsutures and aerosols, and may contain appropriate conventionaladditives, including, for example, preservatives, solvents to assistdrug penetration, and emollients in ointments and creams. Such topicalformulations may also contain compatible conventional carriers, forexample cream or ointment bases, and ethanol or oleyl alcohol forlotions. Such carriers may constitute from about 1% to about 98% byweight of the formulation; more usually they will constitute up to about80% by weight of the formulation.

[0126] For administration to mammals, and particularly humans, it isexpected that the daily dosage level of the active agent will be from0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

[0127] In-dwelling devices include surgical implants, prosthetic devicesand catheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

[0128] The composition of the invention may be administered by injectionto achieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

[0129] Many orthopaedic surgeons consider that humans with prostheticjoints should be considered for antibiotic prophylaxis before dentaltreatment that could produce a bacteremia. Late deep infection is aserious complication sometimes leading to loss of the prosthetic jointand is accompanied by significant morbidity and mortality. It maytherefore be possible to extend the use of the active agent as areplacement for prophylactic antibiotics in this situation.

[0130] In addition to the therapy described above, the compositions ofthis invention may be used generally as a wound treatment agent toprevent adhesion of bacteria to matrix proteins exposed in wound tissueand for prophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

[0131] Alternatively, the composition of the invention may be used tobathe an indwelling device immediately before insertion. The activeagent will preferably be present at a concentration of 1 μg/ml to 10mg/ml for bathing of wounds or indwelling devices.

[0132] A vaccine composition is conveniently in injectable form.Conventional adjuvants may be employed to enhance the immune response. Asuitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, andsuch dose is prteferably administered 1-3 times and with an interval of1-3 weeks. With the indicated dose range, no adverse toxicologicaleffects will be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

[0133] Each reference disclosed herein is incorporated by referenceherein in its entirety. Any patent application to which this applicationclaims priority is also incorporated by reference herein in itsentirety.

[0134] Glossary

[0135] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0136] “Disease(s)” means and disease caused by or related to infectionby a bacteria, including disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

[0137] “Host cell” is a cell which has been transformed or transfected,or is capable of transformation or transfection by an exogenouspolynucleotide sequence.

[0138] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% “identity” to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0139] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

[0140] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including,. for example, simple and complex-cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

[0141] “Polypeptide(s)” refers to any peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide(s)” refers to both short chains, commonlyreferred to as peptides, oligopeptides and oligomers and to longerchains generally referred to as proteins. Polypeptides may contain aminoacids other than the 20 gene encoded amino acids. “Polypeptide(s)”include those modified either by natural processes, such as processingand other post-translational modifications, but also by chemicalmodification techniques. Such modifications are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch-literature, and they are well known to those of skill in theart. It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains, and theamino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selenoylation, sulfation, transfer-RNA mediatedaddition of amino acids to proteins, such as arginylation, andubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993) and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched orcyclic, with or without branching. Cyclic, branched and branchedcircular polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

[0142] “Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

EXAMPLES

[0143] The examples below are carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples are illustrative, butdo not limit the invention.

Example 1 Strain selection, Library Production and Sequencing

[0144] The polynucleotide having a DNA sequence given in Table 1 [SEQ IDNO: 1 or 3] was obtained from a library of clones of chromosomal DNA ofStaphylococcus aureus in E. coli. The sequencing data from two or moreclones containing overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO: 1. Libraries may beprepared by routine methods, for example:

[0145] Methods 1 and 2 Below.

[0146] Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

[0147] Method 1

[0148] Total cellular DNA is mechanically sheared by passage through aneedle in order to size-fractionate according to standard procedures.DNA fragments of up to 11 kbp in size are rendered blunt by treatmentwith exonuclease and DNA polymerase, and EcoRI linkers added. Fragmentsare ligated into the vector Lambda ZapII that has been cut with EcoRI,the library packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

[0149] Method 2

[0150] Total cellular DNA is partially hydrolyzed with a one or acombination of restriction enzymes appropriate to generate a series offragments for cloning into library vectors (e.g., RsaI, PalI, AluI,BshI2351), and such. fragments are size-fractionated according tostandard procedures. EcoRI linkers are ligated to the DNA and thefragments then ligated into the vector Lambda ZapII that have been cutwith EcoRI, the library packaged by standard procedures, and E.coliinfected with the packaged library. The library is amplified by standardprocedures.

Example 2 ribG Characterization

[0151] Riboflavin Biosynthesis Operon

[0152] This ORF is part of an operon which encodes genes ribg, ribB,ribA and ribH. Gene ribG starts at nucleotide 1 and ends at nucleotide1029. Gene ribB starts at nucleotide 1036 and ends at nucleotide 1668.Gene ribA starts at nucleotide 1679 and ends at nucleotide 2860. GeneribH starts at nucleotide 2873 and ends at nucleotide 3337. The operonsequence below is SEQ ID NO:7.ATGGATTATGCGATTCAACTTGCAAATATGGTACAAGGTCAAACAGGTGTTAATCCACCCGTTGGCGCTGTTGTAGTTAATGAAGGTAGGATTGTTGGTATTGGTGCACACTTGAGAAAAGGTGACAAGCATGCGGAGGTTCAAGCACTTGATATGGCACAACAAAATGCTGAAGGTGCGACGATTTATATTACGTTAGAGCCATGTAGTCATTTTGGTTCAACACCACCCTGTGTTAACAAAATTATTGATTGTAAGATAGCAAAAGTAGTATACGCAACAAAAGACAATTCGTTAGACACACATGGTGATGAGACGTTACGGGCTCACGGTATTGAGGTTGAATGCGTTGATGATGAACGGGCATCACAATTATACCAAGACTTTTTTAAAGCAAAAGCAAAGCAACTGCCACAAATTACAGTGAAAGTATCTGCAAGTTTAGATGGTAAACAAGCGAATGATAATGGACAAAGTCAATGGATTACTAACAAAGAGGTTAAACAAGATGTCTATAAGTTAAGACATCGACACGACGCAGTGTTAACTGGAAGACGTACAGTTGAATTAGATGATCCACAATATACTACACGTATTCAAGATGGAAAAAACCCTATAAAAGTAATATTGTCTAAGTCTGGGAATATTCATTTTAATCAGCAAATTTATCAAGATGAATCAACACCAATTTGGATATATACTGAAAATCCAAATTTAACAAGCAATCAAACACATATTGAAATTATTTACTTGAAGTCTTGTGATTTAACAACAATTCTTCACAATTTATATAAAAGAGGAGTTGGAACTTTGCTAGTCGAGGCAGGTCCAACCACTACTTCAGAATTCTCCATCTATTATATAGATGAATTTATTCTCTATTATGCCCCGAAATTAATTGGCGGATCTGGAAATTATCAATTTTATCAAACAAATGATGTGATTGAGATACCAGATGCGAACCAATTTGAAATTGTTCATTCCGAGTTATTAAATCAAAATGTTAAATTAACTTTACGAAAGAAGTGATGATGCATGTTTACTGGCATCGTTGAAGAAATAGGTGTCGTTAAAAGTGTTCAAATTCGTCAATCAGTCAGGACGATTGAAATCGAAGCACATAAGATTACGGCAGATATGCATATTGGTGATTCCATCAGTGTTAATGGTGCATGTTTAACAGTGATTGATTTTGATCAGACATCTTTTACTGTTCAAGTAATTAAAAGCACTGAAAATAAAACCTATTTAGCAGATGTTAAGCGACAATCAGAAGTAAATTTAGAGCGTGCCATGAGTGGTAACGGTAGGTTTGGTGGACATTTTGTCCTCGGTCATGTAGATGAACTAGGAACAGTTTCAAAAATAAATGAAACAGCCAATGCCAAAATTATTACGATTCAATGTAGCCAACATATTAATAATCAGTTAGTTAAGCAAGGGTCTATTACTGTGGATGGTGTAAGTCTAACGGTATTTGATAAGCATGATAACAGTTTTGACATTCATCTTATTCCAGAAACGAGGCGTTCAACGATTTTATCATCCAAAAAATTAGGAGATAAAGTACATTTAGAAACAGACGTTTTGTTTAAATATGTTGAAAATATTTTAAATAAAGATAAAGACCAATTATCTGTAGATAAATTAAGAGCATTTGGTTTTTAGGAGGGGTAGCATGCAATTCGATAATATTGACAGTGCTTTAATGGCTTTAAAAAATGGAGAAACAATTATTGTAGTAGATGATGAGAATCGTGAAAATGAAGGTGATTTAGTAGCGGTTACTGAATGGATGAACGATAATACCATTAATTTTATGGCGAAAGAAGCAAGGGGATTAATATGCGCACCAGTGTCTAAAGATATTGCACAACGTTTGGATTTGGTACAAATGGTTGATGATAACTCCGACATCTTTGGTACGCAATTTACAGTGAGTATTGATCATGTAGATACAACAACAGGAATTAGTGCTTATGAACGTACATTGACTGCCAAAAAGCTCATTGATCCTAGTAGTGAAGCTAAAGATTTTAATCGTCCTGGTCATTTATTTCCATTAGTAGCACAAGATAAAGGCGTATTAGCTAGAAATGGACACACAGAAGCGGCTGTTGATTTAGCTAAACTTACTGGTGCCAAGCCCGCTGGTGTCATTTGTGAGATTATGAATGATGACGGCACGATGGCGAAAGGACAAGATTTACAAAATTTTAAAGAAAAACATCAATTAAAGATGATTACGATTGATGATTTAATTGAATATCGTAAAAAATTAGAACCAGAAATTGAATTTAAGGCAAAAGTGAAAATGCCTACAGATTTCGGAACATTTGATATGTATGGTTTTAAAGCGACATACACAGATGAAGAGATAGTTGTACTGACAAAAGGTGCAATTCGACAACATGAAAATGTACGCTTACATTCTGCGTGCCTTACAGGCGATATTTTCCATAGTCAACGTTGTGATTGTGGTGCTCAACTTGAATCGTCTATGAAGTATATCAATGAACATGGTGGCATGATTATTTATCTACCTCAAGAAGGTCGTGGCATAGGATTGTTAAACAAATTACGCGCATATGAATTAATTGAGCAAGGATATGATACAGTAACTGCAAATTTAGCATTAGGTTTTGATGAAGATTTACGAGATTATCATATTGCTGCACAGATTTTAAAATATTTTAACATCGAACATATCAATTTATTAAGTAATAATCCAAGTAAATTTGAGGGATTAAAACAATATGGCATTGATATTGCAGAAAGAATTGAAGTTATCGTACCAGAAACGGTACATAATCATGATTATATGGTAACGAAAAAAATAAAAATGGGTCATTTAATATAGGAGGACTTTAACATGAATTTTGAAGGTAAATTAATTGGAAAAGATTTGAAAGTTGCAATCGTAGTTAGTCGATTTAATGATTTTATCACTGGAAGATTACTTGAAGGTGCAAAAGATACTTTGATTCGACATGATGTTAATGAAGACAATATTGATGTAGCATTTGTTCCTGGTGCGTTTGAAATTCCTTTAGTAGCTAAAAAATTAGCCTCATCAGGAAATTATGATGCAATAATTACATTAGGATGCGTAATTCGCGGTGCTACGTCTCATTATGATTATGTTTGTAATGAAGTGCGAAAGGTGTTTCTAAAGTAAATGATCAAACTAATGTACCAGTCATATTTGGTATTTTAACGACTGAAAGTATTGAACAAGCTGTGGAAAGAGCAGGTACGAAAGCTGGTAATAAAGGTGCCGAAGCAGCAGTAAGTGCAATTGAAATGGCTAATTTATTAAAATCTATAAAAGCATAG

[0153]

1 7 1029 base pairs nucleic acid double linear 1 ATGGATTATG CGATTCAACTTGCAAATATG GTACAAGGTC AAACAGGTGT TAATCCACCC 60 GTTGGCGCTG TTGTAGTTAATGAAGGTAGG ATTGTTGGTA TTGGTGCACA CTTGAGAAAA 120 GGTGACAAGC ATGCGGAGGTTCAAGCACTT GATATGGCAC AACAAAATGC TGAAGGTGCG 180 ACGATTTATA TTACGTTAGAGCCATGTAGT CATTTTGGTT CAACACCACC CTGTGTTAAC 240 AAAATTATTG ATTGTAAGATAGCAAAAGTA GTATACGCAA CAAAAGACAA TTCGTTAGAC 300 ACACATGGTG ATGAGACGTTACGGGCTCAC GGTATTGAGG TTGAATGCGT TGATGATGAA 360 CGGGCATCAC AATTATACCAAGACTTTTTT AAAGCAAAAG CAAAGCAACT GCCACAAATT 420 ACAGTGAAAG TATCTGCAAGTTTAGATGGT AAACAAGCGA ATGATAATGG ACAAAGTCAA 480 TGGATTACTA ACAAAGAGGTTAAACAAGAT GTCTATAAGT TAAGACATCG ACACGACGCA 540 GTGTTAACTG GAAGACGTACAGTTGAATTA GATGATCCAC AATATACTAC ACGTATTCAA 600 GATGGAAAAA ACCCTATAAAAGTAATATTG TCTAAGTCTG GGAATATTCA TTTTAATCAG 660 CAAATTTATC AAGATGAATCAACACCAATT TGGATATATA CTGAAAATCC AAATTTAACA 720 AGCAATCAAA CACATATTGAAATTATTTAC TTGAAGTCTT GTGATTTAAC AACAATTCTT 780 CACAATTTAT ATAAAAGAGGAGTTGGAACT TTGCTAGTCG AGGCAGGTCC AACCACTACT 840 TCAGAATTCT CCATCTATTATATAGATGAA TTTATTCTCT ATTATGCCCC GAAATTAATT 900 GGCGGATCTG GAAATTATCAATTTTATCAA ACAAATGATG TGATTGAGAT ACCAGATGCG 960 AACCAATTTG AAATTGTTCATTCCGAGTTA TTAAATCAAA ATGTTAAATT AACTTTACGA 1020 AAGAAGTGA 1029 342amino acids amino acid single linear 2 Met Asp Tyr Ala Ile Gln Leu AlaAsn Met Val Gln Gly Gln Thr Gly 1 5 10 15 Val Asn Pro Pro Val Gly AlaVal Val Val Asn Glu Gly Arg Ile Val 20 25 30 Gly Ile Gly Ala His Leu ArgLys Gly Asp Lys His Ala Glu Val Gln 35 40 45 Ala Leu Asp Met Ala Gln GlnAsn Ala Glu Gly Ala Thr Ile Tyr Ile 50 55 60 Thr Leu Glu Pro Cys Ser HisPhe Gly Ser Thr Pro Pro Cys Val Asn 65 70 75 80 Lys Ile Ile Asp Cys LysIle Ala Lys Val Val Tyr Ala Thr Lys Asp 85 90 95 Asn Ser Leu Asp Thr HisGly Asp Glu Thr Leu Arg Ala His Gly Ile 100 105 110 Glu Val Glu Cys ValAsp Asp Glu Arg Ala Ser Gln Leu Tyr Gln Asp 115 120 125 Phe Phe Lys AlaLys Ala Lys Gln Leu Pro Gln Ile Thr Val Lys Val 130 135 140 Ser Ala SerLeu Asp Gly Lys Gln Ala Asn Asp Asn Gly Gln Ser Gln 145 150 155 160 TrpIle Thr Asn Lys Glu Val Lys Gln Asp Val Tyr Lys Leu Arg His 165 170 175Arg His Asp Ala Val Leu Thr Gly Arg Arg Thr Val Glu Leu Asp Asp 180 185190 Pro Gln Tyr Thr Thr Arg Ile Gln Asp Gly Lys Asn Pro Ile Lys Val 195200 205 Ile Leu Ser Lys Ser Gly Asn Ile His Phe Asn Gln Gln Ile Tyr Gln210 215 220 Asp Glu Ser Thr Pro Ile Trp Ile Tyr Thr Glu Asn Pro Asn LeuThr 225 230 235 240 Ser Asn Gln Thr His Ile Glu Ile Ile Tyr Leu Lys SerCys Asp Leu 245 250 255 Thr Thr Ile Leu His Asn Leu Tyr Lys Arg Gly ValGly Thr Leu Leu 260 265 270 Val Glu Ala Gly Pro Thr Thr Thr Ser Glu PheSer Ile Tyr Tyr Ile 275 280 285 Asp Glu Phe Ile Leu Tyr Tyr Ala Pro LysLeu Ile Gly Gly Ser Gly 290 295 300 Asn Tyr Gln Phe Tyr Gln Thr Asn AspVal Ile Glu Ile Pro Asp Ala 305 310 315 320 Asn Gln Phe Glu Ile Val HisSer Glu Leu Leu Asn Gln Asn Val Lys 325 330 335 Leu Thr Leu Arg Lys Lys340 1269 base pairs nucleic acid double linear 3 AANCACCAAT CCNATTGGGAGGNAATCCAA ATCAATNCCC GGANNCCCAA TCCAAGTTAA 60 TTAAGTCCAA GGTTTTGGAACATTACCAAA TATGATTCCG ATGAGGTCAA ATGNCAANCG 120 GTGTTAATAA ACTACGAAATGNTGTGNAAA TGATAGTAGA NCAAGTTGCG CATACAGTNT 180 CTCNATTATA TGATGCTTTAGAATCGAATG AGCAACAACA GCGCAGTTAC AATCAATAAT 240 TTGTAACTAG AAGATAATAAAGAGAACGCT CTATAGAGAC GAATTGAAGG TTTGATTTTA 300 ATGTCTGTTA GTAAGAATCATATCAATGAG ATGCCTATAG TACTCAGATT ATATTAAATT 360 AAAACCGTCA TTAATTGTTTTTTTAGAAAA CATATAGTAT CATTTTAAAT GTAGTTGACA 420 TACTACGTAC TCAAATAATCTATAACAATT TCATATATAA TTCTTTCGGG GCAGGGTGAA 480 ATTCCCAACC GGCAGTAAATAAAGCCTGCG ACCTGCTAAT ATGTTTCATA TTAGTGGCTG 540 ATCTAGTGAG ATTCTAGAGCCGACAGTTAA AGTCTGGATG GGAGAAAGAA TGTTAATTAT 600 CGACAAAGAT AATGTAGCGTATTTGTAAAA ATGTGTACAA ATAGGCTTAT TTAACGATAA 660 ATTTTTCTCC TTTGCATCTTAATTCATGAT GTGAGGATTT TTTGTTTATA GAGGTGATCA 720 TTTGAGTCAA TTTATGGATTATGCGATTCA ACTTCCAAAT ATGGTACAAG GTCANACAGG 780 TGTTAATCCA CCCGTTGGCGCTGTTGTAGT TAATGAAGGT AGGATTGTTG GTATTGGTGC 840 ACACTTGAGA AAAGGTGACAAGCATGCGGA GGTTCAAGCA CTTGATATGG CACAACANAA 900 TGCTGAAGGT GCGACGATTTATATTACGTT AGAGCCATGT AGTCATTTTG GTTCAACACC 960 ACCCTGTGTT AACAAAATTATTGATTGTAA GATAGCANAA GTAGTATTAC NCAACANAAG 1020 ACAATTCCGT TAGACACACATGGGTGATGA GACGTTACGG GGCTCCACGG TATTTGAGGG 1080 TTGAATTGCG TTGGATGATGAACGGGCATC ACAATTATAC CAAAGACTTT TTTTAAAGCA 1140 AAAAGCAAAG CAACTTGCCACAAAATTACA GTGAAAGTNT CTTGAAAGTT TAGATGGGTA 1200 AACAAAGCGA ATTGATAATGGACAAAGTCA ATGGATTACT AACAAAGAGG TTAAACAAGA 1260 TGTCTATAG 1269 99 aminoacids amino acid single linear 4 Met Asp Tyr Ala Ile Gln Leu Pro Asn MetVal Gln Gly Xaa Thr Gly 1 5 10 15 Val Asn Pro Pro Val Gly Ala Val ValVal Asn Glu Gly Arg Ile Val 20 25 30 Gly Ile Gly Ala His Leu Arg Lys GlyAsp Lys His Ala Glu Val Gln 35 40 45 Ala Leu Asp Met Ala Gln Xaa Asn AlaGlu Gly Ala Thr Ile Tyr Ile 50 55 60 Thr Leu Glu Pro Cys Ser His Phe GlySer Thr Pro Pro Cys Val Asn 65 70 75 80 Lys Ile Ile Asp Cys Lys Ile AlaXaa Val Val Leu Xaa Asn Xaa Arg 85 90 95 Gln Phe Arg 21 base pairsnucleic acid single linear 5 ATGAAGGTAG GATTGTTGGT A 21 22 base pairsnucleic acid single linear 6 AGTCTTGGTA TAATTGTGAT GC 22 3336 base pairsnucleic acid double linear 7 ATGGATTATG CGATTCAACT TGCAAATATG GTACAAGGTCAAACAGGTGT TAATCCACCC 60 GTTGGCGCTG TTGTAGTTAA TGAAGGTAGG ATTGTTGGTATTGGTGCACA CTTGAGAAAA 120 GGTGACAAGC ATGCGGAGGT TCAAGCACTT GATATGGCACAACAAAATGC TGAAGGTGCG 180 ACGATTTATA TTACGTTAGA GCCATGTAGT CATTTTGGTTCAACACCACC CTGTGTTAAC 240 AAAATTATTG ATTGTAAGAT AGCAAAAGTA GTATACGCAACAAAAGACAA TTCGTTAGAC 300 ACACATGGTG ATGAGACGTT ACGGGCTCAC GGTATTGAGGTTGAATGCGT TGATGATGAA 360 CGGGCATCAC AATTATACCA AGACTTTTTT AAAGCAAAAGCAAAGCAACT GCCACAAATT 420 ACAGTGAAAG TATCTGCAAG TTTAGATGGT AAACAAGCGAATGATAATGG ACAAAGTCAA 480 TGGATTACTA ACAAAGAGGT TAAACAAGAT GTCTATAAGTTAAGACATCG ACACGACGCA 540 GTGTTAACTG GAAGACGTAC AGTTGAATTA GATGATCCACAATATACTAC ACGTATTCAA 600 GATGGAAAAA ACCCTATAAA AGTAATATTG TCTAAGTCTGGGAATATTCA TTTTAATCAG 660 CAAATTTATC AAGATGAATC AACACCAATT TGGATATATACTGAAAATCC AAATTTAACA 720 AGCAATCAAA CACATATTGA AATTATTTAC TTGAAGTCTTGTGATTTAAC AACAATTCTT 780 CACAATTTAT ATAAAAGAGG AGTTGGAACT TTGCTAGTCGAGGCAGGTCC AACCACTACT 840 TCAGAATTCT CCATCTATTA TATAGATGAA TTTATTCTCTATTATGCCCC GAAATTAATT 900 GGCGGATCTG GAAATTATCA ATTTTATCAA ACAAATGATGTGATTGAGAT ACCAGATGCG 960 AACCAATTTG AAATTGTTCA TTCCGAGTTA TTAAATCAAAATGTTAAATT AACTTTACGA 1020 AAGAAGTGAT GATGCATGTT TACTGGCATC GTTGAAGAAATAGGTGTCGT TAAAAGTGTT 1080 CAAATTCGTC AATCAGTCAG GACGATTGAA ATCGAAGCACATAAGATTAC GGCAGATATG 1140 CATATTGGTG ATTCCATCAG TGTTAATGGT GCATGTTTAACAGTGATTGA TTTTGATCAG 1200 ACATCTTTTA CTGTTCAAGT AATTAAAAGC ACTGAAAATAAAACCTATTT AGCAGATGTT 1260 AAGCGACAAT CAGAAGTAAA TTTAGAGCGT GCCATGAGTGGTAACGGTAG GTTTGGTGGA 1320 CATTTTGTCC TCGGTCATGT AGATGAACTA GGAACAGTTTCAAAAATAAA TGAAACAGCC 1380 AATGCCAAAA TTATTACGAT TCAATGTAGC CAACATATTAATAATCAGTT AGTTAAGCAA 1440 GGGTCTATTA CTGTGGATGG TGTAAGTCTA ACGGTATTTGATAAGCATGA TAACAGTTTT 1500 GACATTCATC TTATTCCAGA AACGAGGCGT TCAACGATTTTATCATCCAA AAAATTAGGA 1560 GATAAAGTAC ATTTAGAAAC AGACGTTTTG TTTAAATATGTTGAAAATAT TTTAAATAAA 1620 GATAAAGACC AATTATCTGT AGATAAATTA AGAGCATTTGGTTTTTAGGA GGGGTAGCAT 1680 GCAATTCGAT AATATTGACA GTGCTTTAAT GGCTTTAAAAAATGGAGAAA CAATTATTGT 1740 AGTAGATGAT GAGAATCGTG AAAATGAAGG TGATTTAGTAGCGGTTACTG AATGGATGAA 1800 CGATAATACC ATTAATTTTA TGGCGAAAGA AGCAAGGGGATTAATATGCG CACCAGTGTC 1860 TAAAGATATT GCACAACGTT TGGATTTGGT ACAAATGGTTGATGATAACT CCGACATCTT 1920 TGGTACGCAA TTTACAGTGA GTATTGATCA TGTAGATACAACAACAGGAA TTAGTGCTTA 1980 TGAACGTACA TTGACTGCCA AAAAGCTCAT TGATCCTAGTAGTGAAGCTA AAGATTTTAA 2040 TCGTCCTGGT CATTTATTTC CATTAGTAGC ACAAGATAAAGGCGTATTAG CTAGAAATGG 2100 ACACACAGAA GCGGCTGTTG ATTTAGCTAA ACTTACTGGTGCCAAGCCCG CTGGTGTCAT 2160 TTGTGAGATT ATGAATGATG ACGGCACGAT GGCGAAAGGACAAGATTTAC AAAATTTTAA 2220 AGAAAAACAT CAATTAAAGA TGATTACGAT TGATGATTTAATTGAATATC GTAAAAAATT 2280 AGAACCAGAA ATTGAATTTA AGGCAAAAGT GAAAATGCCTACAGATTTCG GAACATTTGA 2340 TATGTATGGT TTTAAAGCGA CATACACAGA TGAAGAGATAGTTGTACTGA CAAAAGGTGC 2400 AATTCGACAA CATGAAAATG TACGCTTACA TTCTGCGTGCCTTACAGGCG ATATTTTCCA 2460 TAGTCAACGT TGTGATTGTG GTGCTCAACT TGAATCGTCTATGAAGTATA TCAATGAACA 2520 TGGTGGCATG ATTATTTATC TACCTCAAGA AGGTCGTGGCATAGGATTGT TAAACAAATT 2580 ACGCGCATAT GAATTAATTG AGCAAGGATA TGATACAGTAACTGCAAATT TAGCATTAGG 2640 TTTTGATGAA GATTTACGAG ATTATCATAT TGCTGCACAGATTTTAAAAT ATTTTAACAT 2700 CGAACATATC AATTTATTAA GTAATAATCC AAGTAAATTTGAGGGATTAA AACAATATGG 2760 CATTGATATT GCAGAAAGAA TTGAAGTTAT CGTACCAGAAACGGTACATA ATCATGATTA 2820 TATGGTAACG AAAAAAATAA AAATGGGTCA TTTAATATAGGAGGACTTTA ACATGAATTT 2880 TGAAGGTAAA TTAATTGGAA AAGATTTGAA AGTTGCAATCGTAGTTAGTC GATTTAATGA 2940 TTTTATCACT GGAAGATTAC TTGAAGGTGC AAAAGATACTTTGATTCGAC ATGATGTTAA 3000 TGAAGACAAT ATTGATGTAG CATTTGTTCC TGGTGCGTTTGAAATTCCTT TAGTAGCTAA 3060 AAAATTAGCC TCATCAGGAA ATTATGATGC AATAATTACATTAGGATGCG TAATTCGCGG 3120 TGCTACGTCT CATTATGATT ATGTTTGTAA TGAAGTGCGAAAGGTGTTTC TAAAGTAAAT 3180 GATCAAACTA ATGTACCAGT CATATTTGGT ATTTTAACGACTGAAAGTAT TGAACAAGCT 3240 GTGGAAAGAG CAGGTACGAA AGCTGGTAAT AAAGGTGCCGAAGCAGCAGT AAGTGCAATT 3300 GAAATGGCTA ATTTATTAAA ATCTATAAAA GCATAG 3336

1. An isolated polynucleotide segment comprising: a first polynucleotidesequence, or the full complement of the entire length of the firstpolynucleotide sequence, wherein the first polynucleotide sequence isselected from the group consisting of: (a) a polynucleotide consistingof SEQ ID NO: 1; and (b) a nucleic acid sequence identical to thepolynucleotide of (a) except that, over the entire length correspondingto the polynucleotide of (a), up to thirty nucleotides are substituted,deleted or inserted for every 100 nucleotides of the polynucleotide of(a).
 2. The isolated polynucleotide segment of claim 1, wherein thefirst polynucleotide sequence is selected from the group consisting of:the polynucleotide of (a); and, a nucleic acid sequence identical to thepolynucleotide of (a) except that, over the entire length correspondingto the polynucleotide of (a), up to ten nucleotides are substituted,deleted or inserted for every 100 nucleotides of the polynucleotide of(a).
 3. The isolated polynucleotide segment of claim 1, wherein thefirst polynucleotide sequence is selected from the group consisting of:the polynucleotide of (a); and, a nucleic acid sequence identical to thepolynucleotide of (a) except that, over the entire length correspondingto the polynucleotide of (a), up to five nucleotides are substituted,deleted or inserted for every 100 nucleotides of the polynucleotide of(a).
 4. A vector comprising the isolated polynucleotide segment ofclaim
 1. 5. An isolated host cell comprising the vector of claim
 4. 6.An isolated polynucleotide segment, comprising a first polynucleotidesequence, of the full complement of the entire length of the firstpolynucleotide sequence, wherein the first polynucleotide sequence isselected from the group consisting of: (a) a polynucleotide whichencodes the same mature polypeptide, expressed by the ribG geneexpressed by a polynucleotide comprising SEQ ID NO: 1 contained inStaphylococcus aureus WCUH 29 contained in NCIMB Deposit No. 40771; and,(b) a nucleic acid sequence identical to the polynucleotide of (a)except that, over the entire length corresponding to the polynucleotideof (a), up to five nucleotides are substituted, deleted or inserted forevery 100 nucleotides of the polynucleotide of (a).
 7. The isolatedpolynucleotide segment of claim 6, wherein the first polynucleotidesequence is selected from the group consisting of: the polynucleotide of(a); and, a nucleic acid sequence identical to the polynucleotide of (a)except that, over the entire length corresponding to the polynucleotideof (a), up to three nucleotide are substituted, deleted or inserted forevery 100 nucleotides of the polynucleotide of (a).
 8. An isolatedpolynucleotide segment, comprising a first polynucleotide sequence orthe full complement of the entire length of the first polynucleotidesequence, wherein the first polynucleotide sequence hybridizes to thefull complement of SEQ ID NO: 1, wherein the hybridization conditionsinclude incubation at 42° C. in a solution comprising: 50% formamide,5×SSC (150 mM NaCL, 15 mM trisodium citrate), 50 mM sodium phosphate(pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20micrograms/ml denatured, sheared salmon sperm DNA, followed by washingin 0.1×SSC at about 65° C.
 9. The isolated polynucleotide segment ofclaim 8, wherein the first polynucleotide sequence is identical to SEQID NO: 1 except that, over the entire length corresponding to SEQ ID NO:1, up to five nucleotide are substituted, deleted or inserted for every100 nucleotides of SEQ ID NO:
 1. 10. The isolated polynucleotide segmentof claim 8, wherein the first polynucleotide sequence is identical toSEQ ID NO: 1 except that, over the entire length corresponding to SEQ IDNO: 1, up to three nucleotide are substituted, deleted or inserted forevery 100 nucleotides of SEQ ID NO:
 1. 11. An isolated polynucleotidesegment, comprising a first polynucleotide sequence or the fullcomplement of the entire length of the first polynucleotide sequence,wherein the first polynucleotide sequence is selected from the groupconsisting of: (a) a polynucleotide which encodes a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:2; and, (b) anucleic acid sequence identical to the polynucleotide of (a) exceptthat, over the entire length corresponding to the polynucleotide of (a),up to five nucleotides are substituted, deleted or inserted for every100 nucleotides of the polynucleotide of (a).
 12. A vector comprisingthe isolated polynucleotide segment of claim
 11. 13. An isolated hostcell comprising the vector of claim
 12. 14. The isolated polynucleotidesegment of claim 11, wherein the first polynucleotide sequence isselected from the group consisting of: (a) a polynucleotide whichencodes a polypeptide consisting of the amino acid sequence set forth inSEQ ID NO:2; and, (b) a nucleoice acid sequence identical to thepolynucleotide of (a) except that, over the entire length correspondingto the polynucleotide of (a), up to five nucleoitdes are substituted,deleted or inserted for every 100 nucleotides of the polynucleotide of(a).
 15. A vector comprising the isolated polynucleotide segment ofclaim
 14. 16. An isolated host cell comprising the vector of claim 15.17. A composition comprising the isolated polynucleotide segment ofclaim 1, which isolated polynucleotide segment is according to theformula: X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y wherein, at the 5′ end of themolecule, X is hydrogen, and at the 3′ end of the molecule, Y ishydrogen or a metal, R₁ and R₃ are any nucleic acid residue, m is aninteger between I and 3000 or zero, n is an integer between 1 and 3000or zero, and R₂ is the first polynucleotide sequence.
 18. An isolatedpolynucleotide segment comprising: a first polynucleotide sequence, orthe full complement of the entire length of the first polynucleotidesequence, wherein the first polynucleotide sequence is selected form thegroup consisting of: (a) a polynucleotide encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO:4; (b) a polynucleotideconsisting of SEQ ID NO:3; and, (c) a nucleic acid sequence identical tothe polynucleotide of (b) except that, over the entire lengthcorresponding to the polynucleotide of (b), up to thirty nucleotides aresubstituted, deleted or inserted for every 100 nucleotides of thepolynucleotide of (b).
 19. The isolated polynucleotide segment of claim18, wherein the first polynucleotide sequence is selected from the groupconsisting of: the polynucleotide of (b); and, a nucleic acid sequenceidentical to the polynucleotide of (b) except that, over the entirelength corresponding to the polynucleotide of (b), up to ten nucleotidesare substituted, deleted or inserted for every 100 nucleotides of thepolynucleotide of (b).
 20. The isolated polynucleotide segment of claim18, wherein the first polynucleotide sequence is selected from the groupconsisting of: the polynucleotide of (b); and, a nucleic acid sequenceidentical to the polynucleotide of (b) except that, over the entirelength corresponding to the polynucleotide of (b), up to fivenucleotides are substituted, deleted or inserted for every 100nucleotides of the polynucleotide of (b).